The present invention pertains at least in part to cancer treatment, cancers mediated at least in part by TAK1 and/or other targets, certain chemical compounds, and methods of treating tumors and cancers with the compounds. The present invention also pertains to treating inflammatory and allergic disorders.
In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Among medically important serine/threonine kinases is the family of mitogen-activated protein kinases (MAPKs), which have been shown to function in a wide variety of biological processes (Davis D. J. Trends in Biochem Sci. 19 470-473 (1994); Su B. & Karin M Curr. Opin. Immunol 8 402-411 (1996); Treisman R. Curr. Opin. Cell Biol. 8 205-215 (1996)). MAPKs are activated by phosphorylation on specific tyrosine and threonine residues by MAPK kinases (MAPKKs), which are in turn activated by phosphorylation on serine and serine/threonine residues by MAPKK kinases (MAPKKKs). The MAPKKK family comprises several members including MEKK1, MEKK3, NIK and ASK1 and Raf. Different mechanisms are involved in the activation of MAPKKKs in response to a variety of extracellular stimuli including cytokines, growth factors and environmental stresses.
Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family and has been shown to play critical roles in signaling pathways stimulated by transforming growth factor-β, interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), lipopolysaccharide, receptor activator of NF-κB ligand where it regulates osteoclast differentiation and activation, and IL-8 (Yamaguchi K et al. Science 270 2008-11 (1995); Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem. 274 10641-10648 (1999); He T. et al. FEBS Lett. 467 160-164 (2000); Lee J. et al. J. Leukoc Biol. 68 909-915 (2000); Mizukami J et al. Mol. Cell. Biol. 22 992-1000 (2002); Wald D. et al. J. Immunol. 31 3747-3754 (2002)). TAK1 regulates both the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades in which it phosphorylates MAPK kinases MKK4 and MKK3/6, respectively (Wang W. et al. J. Biol. Chem. 272 22771-22775 (1997); Moriguchi T. et al. J. Biol. Chem. 271 13675-13679 (1996)). NF-kB factors regulate expression of a variety of genes involved in apoptosis, cell cycle, transformation, immune response, and cell adhesion (Barkett M and Gilmore T D. Oncogene, 18, 6910-6924 (1999). TAK1 regulates the IκB kinase (IKK) signaling pathways, leading to the activation of transcription factors AP-1 and NF-κB (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem. 274 10641-10648 (1999); Takaesu G. et al. J. Mol. Biol. 326 110-115 (2003)). In early embryos of the amphibian Xenopus, TAK1 also participates in mesoderm induction and patterning mediated by bone morphogenetic protein (BMP), which is another transforming growth factor β family ligand (Shibuya H. et al. EMBO J. 17 1019-1028 (1998)). In addition, TAK1 is a negative regulator of the Wnt signaling pathway, in which TAK1 down-regulates transcription regulation mediated by a complex of β-catenin and T-cell factor/lymphoid enhancer factor (Meneghini M. D. et al. Nature 399 793-797 (1999); Ishitani T. et al. Nature 399 798-802 (1999)). The role of TAK1 in TNF-α and IL-1β-induced signaling events is evident from TAK1 RNAi experiments in mammalian cells (Takaesu G. et al. J. Mol. Biol. 326 105-115 (2003)) in which IL-1 and TNF-α induced NF-κB and MAPK activation were both inhibited. Over-expression of kinase dead TAK1 inhibits IL-1 and TNK-induced activation of both JNK/p38 and NF-kB (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem. 274 10641-10648 (1999)). TAK1−/−mouse embryonic fibroblasts have diminished IL-1-induced signaling and are embryonic lethal (E11.5) (S. Akira, personal communication). In adult mouse, TAK1 is activated in the myocardium after pressure overload. Expression of constitutively-active TAK1 in myocardium induced myocardial hypertrophy and heart failure in transgenic mice (Zhang D. et al. Nature Med. 6 556-563 (2000)).
TAK1 is activated by the TAK1 binding protein (TAB1) (Shibuya H et al. Science 272 1179-1182 (1996)) via an association with the N-terminal kinase domain of TAK1. It has been reported that the C-terminal 68 amino acids of TAB1 is sufficient for the association and activation of TAK1 (Shibuya H et al. Science 272 1179-1182 (1996)). However, more recent work indicates that the minimum TAB1 segment required includes only residues 480-495 (Ono K. et al. J. Biol. Chem. 276 24396-24400 (2001); Sakurai H. et al. FEBS Lett 474 141-145 (2000)). Deletion mutants of TAB1 show that the aromatic Phe484 residue is critical for TAK1 binding (Ono K. et al. J. Biol. Chem. 276 24396-24400 (2001)). Autophosphorylation of threonine/serine residues in the kinase activation loop are necessary for TAB1-induced TAK1 activation (Sakurai H. et al. FEBS Lett 474 141-145 (2000); Kishimoto K. et al. J. Biol. Chem. 275 7359-7364 (2000)), Ser192 appears as the most likely candidate since a Ser192Ala mutation shows no kinase activity (Kishimoto K. et al. J. Biol. Chem. 275 7359-7364 (2000)).
Since TAK1 is a key molecule in the pro-inflammatory NF-κB signaling pathway a TAK1 inhibitor would be effective in diseases associated with inflammation and tissue destruction such as rheumatoid arthritis and inflammatory bowel disease (Crohn's), as well as in cellular processes such as stress responses, apoptosis, proliferation and differentiation. Various pro-inflammatory cytokines and endotoxins trigger the kinase activity of endogenous TAK1 (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Irie T et al. FEBS Lett. 467 160-164 (2000); Sakurai H. et al. J. Biol. Chem. 274 10641-10648 (1999)) and the Drosophila homolog of TAK1 was recently identified as an essential molecule for host defense signaling in Drosophila (Vidal S. et al. Genes Dev. 15 1900-1912 (1999)). A naturally occurring inhibitor of TAK1, 5Z-7-oxozeaenol, has been identified with an IC50 value of 8 nM. 5Z-7-oxozeaenol has been shown to be selective for TAK1 within the MAPKKK family and relieves inflammation in a picryl chloride-induced ear swelling mouse model (Ninomiya-Tsuji J. et al. J. Biol. Chem. 278 18485 (2003)).
A potential mechanism of TAK1 mediated survival is driven by the ability of TAK1 to phosphorylate IKK and MKKs ultimately leading to the activation of both NF-kB and AP-1, transcription factors that play a role in cell survival.
Others have reported that the TAB1:TAK1:IKKβ:NF-κB signaling axis forms aberrantly in breast cancer cells, and consequently, enables oncogenic signaling by TGF-β (Neil J et al. Cancer Res. 68 1462 (2008)).
Others have reported that TGF-β signaling contributes to tumor angiogenesis and invasion via a mechanism involving matrix metalloproteinase 9 (MMP9) (Safina A et al. Oncogene 26 p2407 (2007)), and that TAK1 is required for TGFb1-mediated regulation of matrix metalloproteinase-9 and metastasis (Safina A et al. Oncogene 2008; 27(9):1198-12072008). Others have reported that TGF-β signaling can induce an epithelial-to-mesenchymal transition (EMT) and contributes to tumor invasion and progression (Ikushima H et al. Nature Reviews Cancer 10 p415 (2010)) and that TAK1 is required for this process (Neil J et al. Cancer Res. 68 1462 (2008)). Thus, TAK1 has been suggested as providing an opportunity for selective inhibition of pro-oncogenic function of TGF-β.
Others have proposed that the signaling pathways by which MDP-NOD2 and LPS-TLR4 induce the production of IL-1β and TNFα converge at the level of TAK1.
Accordingly, there has been an interest in finding selective inhibitors of TAK1 that are effective as therapeutic agents. A challenge has been to find protein kinase inhibitors that act in a selective manner, targeting only TAK1. Since there are numerous protein kinases that are involved in a variety of cellular responses, non-selective inhibitors may lead to unwanted side effects. In this regard, the three-dimensional structure of the kinase would assist in the rational design of inhibitors. The determination of the amino acid residues in TAK1 binding pockets and the determination of the shape of those binding pockets would allow one to design selective inhibitors that bind favorably to this class of enzymes. The determination of the amino acid residues in TAK1 binding pockets and the determination of the shape of those binding pockets would also allow one to determine the binding of compounds to the binding pockets and to, e.g., design inhibitors that can bind to TAK1.
Others have reported that TAK1 plays a key role in proinflammatory signaling by activating JNK, p38, and NF-κB, suggesting that TAK1 inhibition may be effective in preventing inflammation and tissue destruction promoted by proinflammatory cytokines. Ninomiya-Tsuji et al., J. Bio. Chem., 278, 20, pp. 18485-90 (2003). Inhibitors of p38 have also been proposed for treating inflammatory and allergic disorders. US2009/0124604; US2009/0012079.
The following published documents are also noted: Erdogan M et al. Cancer Res. 68 p6224 (2008); Shih S-C et al. PNAS 100 p15859 (2003); US2006/0074102; US2004/0097485; US2003/0004344.
There is a need for effective therapies for use in proliferative disease, including treatments for primary cancers, prevention of metastatic disease, and targeted therapies, including tyrosine kinase inhibitors, such as TAK1 inhibitors, including selective inhibitors, and for potent, orally bioavailable, and efficacious inhibitors.